Antisense inhibition of laminin-8 expression to inhibit human gliomas

Abstract

Using gene array technology, we observed that an increase of the α4 chain-containing Laminin-8 correlated with poor prognosis for patients with brain gliomas. We established that inhibition of Laminin-8 expression by a new generation of highly specific and stable antisense oligonucleotides (Morpholino™) against chains of Laminin-8 could slow or stop the spread of glioma. This was demonstrated in an in vitro model using human glioblastoma multiforme cell lines M059K and U-87MG co-cultured with normal human brain microvascular endothelial cells (HBMVEC). Using Western blot analysis and immunohistochemistry, we con-firmed that antisense treatment effectively blocked laminin-8 protein synthesis. Antisense oligonucleotides against both α4 and β1 chains of laminin-8 blocked significantly the invasion of co-cultures through Matrigel. The results show that Laminin-8 may not only contribute to glioma progression and recurrence as part of the neovascularization process but also by directly increasing the invasive potential of tumor cells.

1. A method for reducing invasiveness of a human glioma in vitro comprising the step of contacting said glioma with a composition that inhibits expression of Laminin-8 by said glioma, wherein Laminin-8 expression is inhibited by inhibiting the expression of Laminin α4 chain and Laminin β1 chain of said Laminin-8, wherein the composition includes a Laminin α4 antisense polynucleotide comprising the 5′ to 3′ polynucleotide sequence characterized by SEQ ID NO: 1. 
     
     
       2. A method for reducing invasiveness of a human glioma in vitro comprising the step of contacting said glioma with a composition that inhibits expression of Laminin-8 by said glioma, wherein Laminin-8 expression is inhibited by inhibiting the expression of Laminin α4 chain and Larninin β1 chain of said Laminin-8, wherein the composition includes a Laminin β1 antisense polynucleotide comprising the 5′ to 3′ polynucleotide sequence characterized by SEQ ID NO: 3. 
     
     
       3. A method for reducing invasiveness of a human glioma in vitro comprising the step of contacting said glioma with a composition that inhibits expression of Laminin-8 by said glioma, wherein Laminin-8 expression is inhibited by inhibiting the expression of Laminin α4 chain and/or Laminin β1 chain of said Laminin-8, wherein the composition includes both a Laminin α4 antisense polynucleotide and a Laminin β1 antisense polynucleotide, wherein the Laminin β1 antisense polynucleotide includes polynucleotides comprising the 5′ to 3′ sequence characterized by SEQ ID NO: 3 and the Laminin α4 antisense polynucleotide includes polynucleotides comprising the 5′ to 3′ sequence characterized by SEQ ID NO: 1.